Multiple-sample rotor assembly for blood fraction preparation

ABSTRACT

A multiple-sample centrifugal rotor for blood fraction preparation is described. The rotor assembly includes an inner disk-shaped portion defining a circular array of whole blood sample-receiving chambers. Blood samples are statically loaded into the respective chambers through static loading ports. Liquids for washing and hemolyzing the cell fractions are introduced to the chambers following recovery of the plasma fraction by means of a central dynamic distribution port and a multiplicity of distribution passageways extending between the dynamic distribution port and the centrifugal ends of respective chambers. Unloading of blood fractions and washing liquid is accomplished through transfer passageways extending from a point intermediate the centrifugal and centripetal ends of the chambers radially inward and then outward to the periphery of the inner disk-shaped rotor assembly portion. A removable outer rotor portion defining at least one collection chamber for receiving materials discharged from the transfer passageways is nested concentrically about the inner rotor assembly portion.

United States Patent 1 1 Tiffany et a1.

Feb. 4, 1975 MULTIPLE-SAMPLE ROTOR ASSEMBLY FOR BLOOD FRACTIONPREPARATION [75] Inventors: Thomas 0. Tiffany; James C.

Mailen, both of Oak Ridge; Wayne F. Johnson, Loudon; Charles D. Scott;W. Wilson Pitt, Jr., both of Oak Ridge, all of Tenn.

[73] Assignee: The United States of America as represented by the UnitedStates Atomic Energy Commission, Washington, DC.

[22] Filed: Dec. 10, 1973 [21] Appl. No.: 423,381

[52] US. Cl 23/2585, 23/259, 233/26, 233/28 [51] Int. Cl B04b 9/12, B04b11/02 [58] Field of Search 23/258.5, 259, 253 R; 233/26, 28

[56] References Cited UNITED STATES PATENTS 3,291,387 12/1966 Billen233/28 3,744,975 7/1973 Mai1en..... 23/259 3,759,666 9/1973 Hill, Jr..."23/253 X 3,795,451 3/1974 Mailen 23/259 X Primary ExaminerR. E. SerwinAttorney, Agent, or Firm-John A. l-loran; David S. Zachry; Stephen D.Hamel [57] ABSTRACT A multiple-sample centrifugal rotor for bloodfraction preparation is described. The rotor assembly includes an innerdisk-shaped portion defining a circular array of whole bloodsample-receiving chambers. Blood samples are statically loaded into therespective chambers through static loading ports. Liquids for washingand hemolyzing the cell fractions are introduced to the chambersfollowing recovery of the plasma fraction by means of a central dynamicdistribution port and a multiplicity of distribution passagewaysextending between the dynamic distribution port and the centrifugal endsof respective chambers. Unloading of blood fractions and washing liquidis accomplished through transfer passageways extending from a pointintermediate the centrifugal and centripetal ends of the chambersradially inward and then outward to the periphery of the innerdisk-shaped rotor assembly portion. A removable outer rotor portiondefining at least one collection chamber for receiving materialsdischarged from the transfer passageways is nested concentrically aboutthe inner rotor assembly portion.

5 Claims, 4 Drawing Figures PATENTED FEB 3. 884. 089

SHEET 1 U? 2 MULTIPLE-SAMPLE ROTOR ASSEMBLY FOR BLOOD FRACTIONPREPARATION I BACKGROUND OF THE INVENTION The invention described hereinrelates generally to blood fraction preparation systems and moreparticularly to an improved multi-sample rotor assembly suitable forseparating blood into plasma and cell fractions, washing and hemolyzingthe cell fraction, and for separately recovering the plasma, hemolysate,and washed cells. It was made in the course of, or under, a contractwith the U.S. Atomic Energy Commission.

In clinical blood work, it is necessary to separate stabilized bloodsamples into plasma and washed cell fractions before many biochemicaltests of interest can be performed. For example, photometric analysismay be performed on the plasma fraction only since the presence of redblood cells interferes with the desired absorption measurement.

Genetic monitoring programs to determine mutations in man caused byenvironmental conditions such as the presence of ionizing radiation,chemical pollutants, etc., as well as other natural causes require thetaking, preparation, and analysis of very large numbers of blood samplesdue to low mutation rates presently postulated. Present clinicallaboratory blood fraction preparation techniques involve tedious andtimeconsuming operations which wouldmake an effective genetic monitoringprogram impractical, however.

It is, accordingly, a general object of the invention to provide a rotorassembly which is suitable for simultaneously preparing blood fractionsfrom a multiplicity of whole blood samples.

Another, more particular object of the invention is to provide a rotorassembly suitable for separating a multiplicity of blood samples intoplasma and cell fractions, washing and hemolyzing the cell fractions,and separately recovering the plasma, hemolysate, and washed cells.

Other objects of the invention will be apparent upon examination of thefollowing written specification and appended drawings.

SUMMARY OF THE INVENTION In accordance with the invention, a rotorassembly is provided for preparing blood fractions from a multiplicityof whole blood samples. The rotor assembly includes an inner rotorportion defining a circular array of radially extending whole bloodsample-receiving chambers. Static loading ports communicate between thetop end surface of the inner rotor portion and respective chambers inthe circular array of chambers to facilitate the static loading of wholeblood samples therein. A central dynamic distribution port communicates,by way of a multiplicity of radially extending distribution passageways,with the centrifugal ends of each of the sample receiving chambers.Transfer passageways for unloading of blood fractions and washing liquidfrom the chambers extend from a point intermediate the centrifugal andcentripetal ends of the chambers radially inward and then outward to theperiphery of the inner rotor portion. A removable outer rotor portion,defining at least one collection chamber for receiving materialsdischarged from the transfer passageways, is nested concentrically aboutthe inner rotor portion. Rotor assemblies made in accordance with theinvention are suitable for separating blood into plasma and cellfractions, washing and hemolyzing the cell fraction, and for separatelyrecovering the plasma, hemolysate, and washed cells.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view,vertically sectioned, showing a rotor assembly made in accordance withthe invention mounted within a turntable.

FIG. 2 is a top plan view, partially cut away, of the rotor assembly ofFIG. 1.

FIG. 3 is a perspective view of a removable outer rotor portion designedfor collecting cell fraction wash liquid.

FIG. 4 is a perspective view of a removable outer rotor portion definingan array of sample analysis cuvettes suitable for use with the inventionand in a fast analyzer of the rotary cuvette type.

DESCRIPTION OF THE PREFERRED EMBODIMENT Referring now to the drawings,initially to FIG. 1, a rotor assembly made in accordance with theinvention is shown nested within a motor driven turntable 1. As shown,turntable 1 is provided with passageways 2 extending from the turntableaxis to several points about its periphery. Passageways 2 communicatewith a suitable vacuum source for reasons explained below in connectionwith operation of the subject rotor assembly.

The rotor assembly includes an inner disk-shaped rotor portion 3defining a circular array (only one shown in FIG. 1) of whole bloodsample-receiving chambers 4. Static loading ports 5, extending throughthe top surface of disk-shaped rotor portion 3, facilitate the directloading of individual whole blood samples into respectivesample-receiving chambers 4 under static conditions. Ports 5 aredisposed near the centripetal ends of chambers 4 to avoid overflow ofchamber contents through the ports during rotation. Other liquids suchas washing or hemolyzing liquids may be dynamically distributed to theentire array of chambers 4 by means of a central dynamic distributionport 6 and a multiplicity of distribution passageways 7 communicatingbetween that port and the centrifugal ends of respective chambers 4.Distribution passageways 7 intersect at the periphery of dynamicdistribution port 6 to create a saw-tooth or serrated-edge effect whichprovides a substantially equal distribution of liquid into passageways 7when the rotor assembly is rotating and liquid is injected into port 6.Transfer passageways 8 extend from a radially intermediate point alongthe bottom of each chamber 4, radially inward, upward, and then radiallyoutward to the periphery of inner diskshaped rotor portion 3. FIG. 3 iscut away to illustrate a passageway 8.

Nested concentrically about inner disk-shaped rotor portion 3 is aremovable outer rotor portion 9 defining a plurality of collectionchambers 10 for receiving liquids discharged from respectivesample-receiving chambers 4. As shown, chambers 10 open in register withthe radial extremities of respective passageways 8. Vents 11 extendradially inward and upward from each collection chamber 10 to the topsurface of rotor portion 9.

A vacuum annulus 12 is formed above outer rotor portion 9 and theadjoining area of inner rotor portion 3 by means of an annular sealingdisk 13 positioned between upstanding rim 14 of turntable 1 and a raisedflange formed on the top surface of inner rotor portion 3. O-rings 16provide the necessary vacuum seal while permitting removal of disk 13for replacement of outer rotor portion 9 or removal of the entire rotorassembly from the turntable. As shown, passageways 2 open at the side ofannulus 12.

Rotor assemblies made in accordance with the invention are convenientlyfabricated by machining cavities and channels into a central plasticdisk 17 which is sandwiched between and attached, by cementing forexample, to top and bottom cover disks l8 and 19, to form chambers andinterconnecting channels. Although the sandwich construction isspecifically illustrated with reference to the inner rotor portion 3,outer rotor portion 9 may be constructed in a like manner. All or partof the disks may conveniently be made of transparent plastic tofacilitate the observation, using a strobe light for example, of therotor contents.

F IG. 2 is a plan view of a rotor assembly identical to that illustratedin the perspective view of FIG. I. As shown in FIG. 2, a multiplicity ofsample handling systems comprising sample-receiving chambers 4,associated passageways 7 and 8, and collection chambers 10 are containedin a single rotor assembly, thereby facilitating the simultaneouspreparation of separate blood fractions from multiple samples. More orfewer than the eight sample handling systems illustrated may be providedin a single rotor depending on the size of the rotor and the respectivechambers used therein.

FIG. 3 illustrates part of a removable outer rotor portion 9' designedfor collecting cell fraction wash liquid. As shown, a single annularcollection chamber 10 is provided for collecting wash liquid from all ofthe sample handling systems in a rotor assembly. A single annularopening 21 facilitates the discharge of wash liquid from the transferpassageways into collection chamber 10. Intermingling of wash liquidfrom the respective systems is permitted in chamber 10 since the washliquid is discarded without additional analysis except as needed todetermine the need, if any, for additional washing. Vents 11 extend fromchamber 10' to the top surface of rotor portion 9 FIG. 4 illustrates aremovable outer rotor portion 9" defining an array of cuvettes I0"suitable for use in a fast photometric analyzer of the rotary cuvettetype such as described in U.S. Pat. No. 3,744,974 issued to commonassignee on July 10, I973, in the name of W. L. Maddox et al.Transparent top and bottom plates 18" and 19" permit light passagethrough the cuvettes for photometric analysis in accordance with theteachings of that patent. Vents 11" extend radially inward and upwardfrom each cuvette to the top of plate 18".

ROTOR OPERATION Passageways 8 open within chambers 4 at a radiallyintermediate position which is calculated to be slightly centripetal tothe blood cell-plasma interface for normal blood and specific samplevolumes in order to remove most of the plasma fraction withoutdisturbing the blood cell fraction. The rotor is then stopped and theouter rotor portion 9 removed to permit recovery and testing of therespective plasma fractions.

Following recovery of the plasma fractions, outer rotor portion 9 isreplaced with an outer rotor portion 9' such as that shown in FIG. 3 andthe reassembled rotor rotated at about 2,000 rpm. A selected volume ofphysiological saline wash solution is then injected into dynamicdistribution port 6 causing it to pass in essentially equal volumesthrough passageways 7 to respective chambers 4 where it mixes with andwashes the blood cells remaining in those chambers. Mixing of the salinesolution and cells is enhanced by rapid braking Using a rotor assemblyand turntable substantially as I and acceleration of the rotor. Thecells are then centrifugally resedimented and the wash liquid drawn offinto collection chamber 10 by applying vacuum through passageways 2.Vents 11' provide communication between vacuum nnulus l2 and collectonchamber 10, thereby causing reduced pressure in that chamber and theresultant transfer of the saline wash solution from chambers 4 in amanner similar to that used to transfer the plasma fraction tocollection chambers 10. The washing step is repeated as needed toachieve the desired cleansing action.

Following cell washing, the rotor assembly is stopped and outer rotorportion 9 replaced with an outer rotor portion having individualcollection chambers 10 identical to that used in the collection ofplasma fractions. The rotor assembly is then accelerated and lyzingliquid such as distilled water distributed to respective chambers 4 byinjecting it into dynamic distribution port 6 in the same manner as theaforementioned wash solu tion. I-Iemolysate is recovered by (1)dynamically introducing carbon tetrachloride into port 6 to settle celldebris against the centrifugal end of the chambers 4 and tocentripetally displace the hemolysate and (2) applying vacuum throughpassageways 2 so as to cause the lysate to pass through passageways 8 torespective collection chambers 10. Sufficient carbon tetrachloride canbe used to displace the hemolysate to a point where the carbontetrachloride-hemolysate interface is just centrifugal to the opening ofpassageways 8 in chambers 4. Alternatively, recovery can be effected by(l) centrifugally compacting cell debris against the centrifugal end ofchambers 4, (2) bringing the rotor assembly to a standstill, and (3)applying vacuum through passageways 2.

Where it is desired to photometrically analyze the plasma fractions ofthe blood samples, an outer rotor portion defining sample analysiscuvettes as shown in FIG. 4 can be used to collect those fractions. Thecuvettes can be preloaded with reactants and the entire rotor assemblydisposed in a fast analyzer system as referenced above or the outerrotor portion containing the cuvettes transferred to a fast analyzerwhere both reagent addition and photometric analysis functions areperformed. Subsequent washing and lyzing of the cell fraction can becarried out in the manner described above.

Following the above-described washing step, all or part of the washedblood cell fractions may be recovered for testing or storage for futurecomparison. Such recovery is effected with the rotor assembly at rest orat low speed by applying vacuum to passageways 2, thereby causing cellsfilling the bottoms of chambers 4 to pass through passageways 8. Thecells are collected in respective collection chambers in an outer rotorportion identical to that used to collect plasma fractions. Where onlypart of the cell fractions is recovered, the remaining cell fractionscan be lyzed and the lysate recovered in the manner previouslydescribed.

The above description of one embodiment of the invention should not beinterpreted in a limiting sense. For example, the exact configuration ofchambers 4 and associated passageways 7 and 8 may vary from thatillustrated without departing from the invention. Passageways 8 mayextend from the sides rather than the bottoms of chambers 4 if transferof chamber contents under dynamic conditions only is contemplated. It isnecessary, however, that passageway 8 extend radially inward to a pointcentripetal to the maximum centripetal level of sample liquid in chamber4 to avoid overflow of the sample during rotation. Likewise, passageways7 and 8 should extend upward to a level sufficient to prevent overflowof sample liquid from chambers 4 under static conditions. it isintended, rather, that the invention be limited only by the scope of theappended claims.

What is claimed is:

l. A multiple-sample centrifugal rotor assembly for blood fractionpreparation comprising:

a. an inner disk-shaped rotor portion having a top end surface, saidinner rotor portion defining:

i. a multiplicity of radially oriented samplereceiving chambers havingcentripetal and centrifugal ends, said sample-receiving chambers beingdisposed in a circular array;

ii. a multiplicity of static sample-loading ports, each of said portscommunicating between said top end surface and respectivesample-receiving chambers;

iii. a centrally located dynamic distribution port open to said top endsurface;

iv. a multiplicity of distribution passageways, each of saiddistribution passageways communicating between said dynamic distributionport and the centrifugal end of a respective sample-receiving chamber;and

v. a multiplicity of transfer passageways. each of said transferpassageways communicating be tween one of said sample-receiving chambersat a point intermedite its centrifugal and centripetal ends and theradial periphery of said inner rotor portion, each of said transferpassageways extending radially inward from its point of communicationwith said sample-receiving chamber and then generally radially outwardto said radial periphery of said inner rotor portion; and

b. a removable outer rotor portion having an annular configurationnested concentrically 'about said inner rotor portion, said outer rotorportion defining at least one collection chamber in fluid communicationwith said transfer passageways in said inner rotor portion for receivingmaterial discharaged from said transfer passageways.

2. The rotor assembly of claim 1 wherein said removable outer rotorportion defines a multiplicity of collection chambers, each of saidcollection chambers having an opening in register with the radialextremity of a respective transfer passageway so as to receive liquidsdischarged from said transfer passageways.

3. The rotor assembly of claim 2 wherein each of said collectionchambers is vented through the top surface of said outer rotor portion.

4. The rotor assembly of claim 1 wherein said static sample loadingports communicate with the centripetal ends of said sample-receivingchambers.

5. The rotor assembly of claim 1 wherein said transfer passagewayscommunicate with the bottom ends of said sample-receiving chambers.

1. A MULTIPLE-SAMPLE CENTRIFUGAL ROTOR ASSEMBLY FOR BLOOD FRACTIONPREPARATION COMPRISING: A. AN INNER DISK-SHAPED ROTOR PORTION HAVING ATOP END SURFACE, SAID INNER ROTOR PORTION DEFINING: I. A MULTIPLICITY OFRADIALLY ORIENTED SAMPLE-RECEIVING CHAMBERS HAVING CENTRIPETAL ANDCENTRIFUGAL ENDS, SAID SAMPLE-RECEIVING CHAMBERS BEING DISPOSED IN ACIRCULAR ARRAY; II. A MULTIPLICITY OF STATIC SAMPLE-LOADING PORTS, EACHOF SAID PORTS COMMUNICATING BETWEEN SAID TOP END SURFACE AND RESPECTIVESAMPLE-RECEIVING CHAMBERS; III. A CENTRALLY LOCATED DYNAMIC DISTRIBUTIONPORT OPEN TO SAID TOP END SURFACE; IV. A MULTIPLICITY OF DISTRIBUTIONPASSAGEWAYS, EACH OF SAID DISTRIBUTION PASSAGEWAYS COMMUNICATING BETWEENSAID DYNAMIC DISTRIBUTION PORT AND THE CENTRIFUGAL END OF A RESPECTIVESAMPLE-RECEIVING CHAMBER; AND V. A MULTIPLICITY OF TRANSFER PASSAGEWAYS,EACH OF SAID TRANSFER PASSAGEWAYS COMMUNICATING BETWEEN ONE OF SAIDSAMPLE-RECEIVING CHAMBERS AT A POINT INTERMEDITE ITS CENTRIFUGAL ANDCENTRIPETAL ENDS AND THE RADIAL PERIPHERY OF SAID INNER ROTOR PORTION,EACH OF SAID TRANSFER PASSAGEWAYS EXTENDING RADIALLY INWARD FROM ITSPOINT OF COMMUNICATION WITH SAID SAMPLE-RECEIVING CHAMBER AND THENGENERALLY RADIALLY OUTWARD TO SAID RADIAL PERIPHERY OF SAID INNER ROTORPORTION; AND B. A REMOVABLE OUTER ROTOR PORTION HAVING AN ANNULARCONFIGURATION NESTED CONCENTRICALLY ABOUT SAID INNER ROTOR PORTION, SAIDOUTER ROTOR PORTION DEFINING AT LEAST ONE COLLECTION CHAMBER IN FLUIDCOMMUNICATION WITH SAID TRANSFER PASSAGEWAYS IN SAID INNER ROTOR PORTIONFOR RECEIVING MATERIAL DISCHARAGED FROM SAID TRANSFER PASSAGEWAYS. 2.The rotor assembly of claim 1 wherein said removable outer rotor portiondefines a multiplicity of collection chambers, each of said collectionchambers having an opening in register with the radial extremity of arespective transfer passageway so as to receive liquids discharged fromsaid transfer passageways.
 3. The rotor assembly of claim 2 wherein eachof said collection chambers is vented through the top surface of saidouter rotor portion.
 4. The rotor assembly of claim 1 wherein saidstatic sample loading ports communicate with the centripetal ends ofsaid sample-receiving chambers.
 5. The rotor assembly of claim 1 whereinsaid transfer passageways communicate with the bottom ends of saidsample-receiving chambers.